A possible role of the 5'' terminal sequence of 16S ribosomal RNA in the recognition of initiation sequences for protein synthesis.

Extensive complementarity is found between the 5' end of 16S ribosomal RNA and protein synthesis initiation sites of bacteriophage RNA. Hybrids can be constructed from base sequences of 16S-RNA and two initiation regions on phage RNA. A model is proposed for the involvement of 16S-RNA in the unfolding of hairpin loops containing the initiation codon AUG.     

A ribonucleoprotein fragment of the 30 S ribosome of E. coli containing two contiguous domains of the 16 S RNA.

Ribonucleoprotein fragments of the 30 S ribosome of E. coli have been prepared by limited ribonuclease digestion and mild heating of the ribosome in a constant ionic environment. One such fragment has been described previously. A second electrophoretically homogeneous fragment has now been isolated and its RNA and protein moieties have been characterized. It contains the 5' half of the 16 S RNA, encompassing domains I and II except for the extreme 5' terminus and several small gaps. Seven proteins are present: S4, S5, S6, S8, S12, S15 and S20. The RNA binding sites of five of these proteins are known, and all are RNA sequences that are present in the fragment. Published neutron scattering and immuno-electron microscopic data indicate that six of the proteins are clustered together in a cross sectional slice through the center of the subunit. After deproteinization, the RNA moiety gives two bands in gel electrophoresis, one containing domains I and II and the other, essentially only domain II. The former, although larger, migrates faster in gel electrophoresis, indicating that RNA domains I and II interact with each other in such a way as to become more compact than domain II by itself.     

RNA--protein interactions in the ribosome. IV. Structure and properties of binding sites for proteins S4, S16/S17 and S20 in the 16S RNA

Proteins S4, S16/S17 and S20 of the 30 S ribosomal subunit of Escherichia coli+ associate with specific binding sites in the 16 S ribosomal RNA. A systematic investigation of the co-operative interactions that occur when two or more of these proteins simultaneously attach to the 16 S RNA indicate that their binding sites lie near to one another. The binding site for S4 has previously been located within a 550-nucleotide RNA fragment of approximately 9 S that arises from the 5′-terminal portion of the 16 S RNA upon limited hydrolysis with pancreatic ribonuclease. The 9 S RNA was unable to associate with S20 and S16/S17, however, either alone or in combination. A fragment of similar size and nucleotide sequence, termed the 9 S¹ RNA, has been isolated following ribonuclease digestion of the complex of 16 S RNA with S20 and S16/S17. The 9 S¹ RNA bound not only S4, but S20 and S16/S17 as well, although the fragment complex was stable only when both of the latter protein fractions were present together. Nonetheless, measurements of binding stoichiometry demonstrated the interactions to be specific under these conditions. A comparison of the 9 S and 9 S¹ RNAs by electrophoresis in polyacrylamide gels containing urea revealed that the two fragments differ substantially in the number and distribution of hidden breaks. Contrary to expectation, the RNA in the ribonucleoprotein complex appeared to be more accessible to ribonuclease than the free 16 S RNA as judged by the smaller average length of the sub-fragments recovered from the 9 S¹ RNA. These results suggest that the binding of S4, S16/S17 and S20 brings about a conformational alteration within the 5′ third of the 16 S RNA.To delineate further the portions of the RNA chain that interact with S4, S16/S17 and S20, specific fragments encompassing subsequences from the 5′ third of the 16 S RNA were sought. Two such fragments, designated 12 S-I and 12 S-II, were purified by polyacrylamide gel electrophoresis from partial T₁ ribonuclease digests of the 16 S RNA. The two RNAs, which contain 290 and 210 nucleotides, respectively, are contiguous and together span the entire 5′-terminal 500 residues of the 16 S RNA molecule. When tested individually, neither 12 S-I nor 12 S-II bound S4, S16/S17 or S20. If heated together at 40 °C in the presence of Mg²⁺ ions, however, the two fragments together formed an 8 S complex which associated with S4 alone, with S16/S17 + S20 in combination, and with S4 + S16/S17 + S20 when incubated with an un fractionated mixture of 30 S subunit proteins. These results imply that each fragment contains part of the corresponding binding sites.     

Characterisation of RNA fragments obtained by mild nuclease digestion of 30-S ribosomal subunits from Escherichia coli.

When Escherichia coli 30-S ribosomal subunits are hydrolysed under mild conditions, two ribonucleoprotein fragments of unequal size are produced. Knowledge of the RNA sequences contained in these hydrolysis products was required for the experiments described in the preceding paper, and the RNA sub-fragments have therefore been examined by oligonucleotide analysis. Two well-defined small fragments of free RNA, produced concomitantly with the ribonucleoprotein fragments, were also analysed. The larger ribonucleoprotein fragment, containing predominantly proteins S4, S5, S8, S15, S16 (17) and S20, contains a complex mixture of RNA sub-fragments varying from about 100 to 800 nucleotides in length. All these fragments arose from the 5'-terminal 900 nucleotides of 16-S RNA, corresponding to the well-known 12-S fragment. No long-range interactions could be detected within this RNA region in these experiments. The RNA from the smaller ribonucleoprotein fragment (containing proteins S7, S9 S10, S14 and S19) has been described in detail previously, and consists of about 450 nucleotides near the 3' end of the 16-S RNA, but lacking the 3'-terminal 150 nucleotides. The two small free RNA fragments (above) partly account for these missing 150 nucleotides; both fragments arose from section A of the 16-S RNA, but section J (the 3'-terminal 50 nucleotides) was not found. This result suggests that the 3' region of 16-S RNA is not involved in stable interactions with protein.     

The distance between S1, S21, and the 3' end of 16S RNA in 30S ribosomal subunits. The effect of poly(uridylic acid) and 50S subunits on these distances

The apparent distances between probes covalently attached to the cysteine thiols of S1 or S21 and the 3' end of 16S RNA in Escherichia coli 30S ribosomal subunits were determined by non-radiative energy transfer to be: S21-16S RNA, 5.1 nm; S21-S1, 6.9 nm; S1-16S RNA, 6.8 nm. Binding of poly(uridylic acid) to 30S subunits causes the apparent distances between S1 and 16S RNA or S21 and 16S RNA to increase by more than 1.2 nm and 0.5 nm, respectively, but has little or no effect on the S1-S21 distance. Binding of 50S subunits causes an apparent increase in the S21-16S RNA and S21-S1 distances by 1.0 nm and 0.8 nm, respectively, but has little or no effect on the S1-16S-RNA distance.     

5S-rRNA-containing ribonucleoproteins from rabbit muscle and liver. Complex and partial primary structures

A 5S-rRNA-containing ribonucleoprotein was purified to homogeneity from a rabbit muscle extract through its affinity to phosphofructokinase-1 and then structurally characterized. This RNP was compared to the 5S-rRNA-containing ribonucleoprotein extracted from rabbit liver ribosomal 60S subunits with EDTA. Analytical gel filtration revealed a molecular mass of 70-80 kDa for both complexes. Gel electrophoresis of the ribosomal complex revealed three protein components, one migrating as a band of 35 kDa and two other small polypeptides of apparently 16.5 kDa and 17.5 kDa. In the sarcoplasmic RNP these small polypeptides were absent. However, besides a major component of 35 kDa, up to five slightly larger and smaller species of 31.5-36.5 kDa were detected. Despite this heterogeneity, only one N-terminal amino acid sequence was obtained for the isolated sarcoplasmic protein, suggesting a C-terminal heterogeneity of one single polypeptide. Within the first 46 amino acid residues no difference between the sequences of the isolated 35-kDa components of sarcoplasmic and ribosomal complexes was found. Homology criteria indicated that this component belongs to the ribosomal protein L5 family. The RNA was identified by complete enzymatic sequencing as 5S rRNA; it was also identical in both complexes and is strongly homologous to 5S rRNA of man. Both L5-5S-RNA complexes could be resolved by hydroxyapatite chromatography into three species still consisting of both protein and RNA. 5'-Terminal dephosphorylation experiments showed that this heterogeneity is exclusively due to the differing number (1-3) of 5'-terminal phosphates. The two additional low-molecular-mass proteins were stably associated to the ribosomal RNP at high salt concentrations in a stoichiometry of about 2:1. They were identified as the acidic phosphoproteins P2/P3 by N-terminal sequencing. High phosphate concentrations facilitated their dissociation from the L5-5S-RNA complex. For the sarcoplasmic L5-5S-RNA complex a hitherto unknown interaction with phosphofructokinase-1, affecting the enzymatic properties, was demonstrated.     

A small angle x-ray scattering study of a fragment derived from E. coli 5S RNA.

The structure of a 62 base nuclease resistant fragment of E. coli 5S RNA (bases 1-11, 69-87, 89-120) has been examined by small angle x-ray scattering. The results obtained are indistinguishable from those expected if this oligonucleotide complex were a perfect RNA double helix of about 30 base pairs. These results indicate that this portion of 5S RNA is in a configuration which is approximately double helical, even though proper base pairing is possible over only half its length.     

Neutron scattering study of the 13 S fragment of 16 S RNA and its complex with ribosomal protein S4

A fragment with a molecular weight of 170,000 and a sedimentation coefficient of 13 S which is capable of specifically binding ribosomal protein S4 has been obtained by digestion of Escherichia coli 16 S RNA with ribonuclease A. The 13 S fragment of 16 S RNA and its complex with protein S4 have been studied by different physical methods; in the first place, by neutron scattering. It has been shown that this fragment is very compact in solution. The radii of gyration of this fragment (50 ± 3 Å) and of protein S4 within the complex (17 ± 3 Å) coincide, within the limits of experimental error, with the radii of gyration for the free RNA fragment (47 ± 2 Å) and the free ribosomal protein S4 in solution (18 ± 2 Å). Hence the conclusion is drawn that the compactness of the RNA fragment and the ribosomal protein does not change on complex formation. The compact 13 S fragment of 16 S RNA is shown to be contrast-matched in solvent containing 70% ²H₂O which corresponds to a value for the partial specific volume of RNA of 0.537 cm³/g.     

Nucleotide Sequence Analyses and Predicted Coding of Bunyavirus Genome RNA Species

We performed 3′ RNA sequence analyses of [³²P]pCp-end-labeled La Crosse (LAC) virus, alternate LAC virus isolate L74, and snowshoe hare bunyavirus large (L), medium (M), and small (S) negative-stranded viral RNA species to determine the coding capabilities of these species. These analyses were confirmed by dideoxy primer extension studies in which we used a synthetic oligodeoxynucleotide primer complementary to the conserved 3′-terminal decanucleotide of the three viral RNA species (Clerx-van Haaster and Bishop, Virology 105:564-574, 1980). The deduced sequences predicted translation of two S-RNA gene products that were read in overlapping reading frames. So far, only single contiguous open reading frames have been identified for the viral M- and L-RNA species. For the negative-stranded M-RNA species of all three viruses, the single reading frame developed from the first 3′-proximal UAC triplet. Likewise, for the L-RNA of the alternate LAC isolate, a single open reading frame developed from the first 3′-proximal UAC triplet. The corresponding L-RNA sequences of prototype LAC and snowshoe hare viruses initiated open reading frames; however, for both viral L-RNA species there was a preceding 3′-proximal UAC triplet in another reading frame that was followed shortly afterward by a termination codon. A comparison of the sequence data obtained for snowshoe hare virus, LAC virus, and the alternate LAC virus isolate showed that the identified nucleotide substitutions were sufficient to account for some of the fingerprint differences in the L-, M-, and S-RNA species of the three viruses. Unlike the distribution of the L- and M-RNA substitutions, significantly fewer nucleotide substitutions occurred after the initial UAC triplet of the S-RNA species than before this triplet, implying that the overlapping genes of the S RNA provided a constraint against evolution by point mutation. The comparative sequence analyses predicted amino acid differences among the corresponding L-, M-, and S-RNA gene products of snowshoe hare virus and the two LAC virus isolates.     

Isolation and physical study of the 13S fragment of 16S RNA and its complex with ribosomal protein S4

A fragment of E. coli 16S RNA has been obtained by its hydrolysis with pancreatic RNAase A coupled to Sepharose 4B. This fragment has a molecular weight of 170 000 and a sedimentation coefficient of 13S. It does not aggregate in solution and binds with the ribosomal protein S4. The 13S fragment and it complex with the protein S4 have been studied by different physical methods in the first place, by neutron scattering. It has been shown that this fragment is compact in solution. The radii of gyration of the fragment (50 +/- 3 A) and of the protein S4 within the complex (17 +/- 3 A) coincide, within limits of experimental error, with the radii of gyration for the free RNA fragment (47 +/- 2 A) and the free ribosomal protein S4 in solution (18 +/- 2 A). Hence, the conclusion is made that the compactness of the 13S fragment of the 16S RNA and the ribosomal protein S4 does not change at the complex formation. The compact 13S fragment of the 16S RNA is shown to be contrast matched in the H2O/D2O mixture containing 70% D2O which corresponds to its partial specific volume v equal to 0.537 cm3/g.     

RNA-protein interactions in the ribosome. II. Binding of ribosomal proteins to isolated fragments of the 16 S RNA

Specific fragments of the 16 S ribosomal RNA of Escherichia coli have been isolated and tested for their ability to interact with proteins of the 30 S ribosomal subunit. The 12 S RNA, a 900-nucleotide fragment derived from the 5′-terminal portion of the 16 S RNA, was shown to form specific complexes with proteins S4, S8, S15, and S20. The stoichiometry of binding at saturation was determined in each case. Interaction between the 12 S RNA and protein fraction $\text{S}\text{16}\text{S}\text{17}$ was detected in the presence of S4, S8, S15 and S20; only these proteins were able to bind to this fragment, even when all 21 proteins of the 30 S subunit were added to the reaction mixture. Protein S4 also interacted specifically with the 9 S RNA, a fragment of 500 nucleotides that corresponds to the 5′-terminal third of the 16 S RNA, and protein S15 bound independently to the 4 S RNA, a fragment containing 140 nucleotides situated toward the middle of the RNA molecule. None of the proteins interacted with the 600-nucleotide 8 S fragment that arose from the 3′-end of the 16 S RNA.When the 16 S RNA was incubated with an unfractionated mixture of 30 S subunit proteins at 0 °C, 10 to 12 of the proteins interacted with the ribosomal RNA to form the reconstitution intermediate (RI) particle. Limited hydrolysis of this particle with T₁ ribonuclease yielded 14 S and 8 S subparticles whose RNA components were indistinguishable from the 12 S and 8 S RNAs isolated from digests of free 16 S RNA. The 14 S subparticle contained proteins S6 and S18 in addition to the RNA-binding proteins S4, S8, S15, S20 and $\text{S}\text{16}\text{S}\text{17}$. The 8 S subparticle contained proteins S7, S9, S13 and S19. These findings serve to localize the sites at which proteins incapable of independent interaction with 16 S RNA are fixed during the early stages of 30 S subunit assembly.     

A ribonucleoprotein fragment of the 30 S ribosome of E. coli: evidence for long range RNA-RNA interactions.

A stable homogeneous ribonucleoprotein fragment of the 30 S ribosomal subunit of E. coli has been prepared by mild nuclease digestion and heating in a constant ionic environment. The fragment contains about half of the 16 S ribosomal RNa and six proteins: S4, S7, S9, S13, S16 and S19. The RNA moiety contains the reported binding sites of all six proteins. After deproteinization, 80% of the RNA migrated as two major electrophoretic bands, which were isolated and sequenced. Each band contained sequences from the 5' and 3' thirds of the 16 S RNA but none from the central third. That these two noncontiguous RNA domains migrated together electrophoretically in Mg++-containing gels after deproteinization constitutes direct evidence that the 16 S RNA is folded in the intact ribosome so as to bring the two domains close together and that there are RNA-RNA interactions between them in the presence of Mg++.     

Specific cleavage of ribosomal RNA caused by alpha sarcin.

Alpha sarcin causes the specific cleavage of RNA from 80S ribosomes and 60S subunits of yeast, but not from the 40S subunits to produce a small RNA fragment. The fragment was also produced on treatment of the 60S subunits of wheat germ ribosomes. The fragment has a molecular weight of 100,000 and is a cleavage product of the large RNA species in the 60S subunits. The fragment is not derived from the 5'end of the yeast 25S RNA nor does it bind to 5.8S RNA and we propose that the fragment represents the 3' terminal 320 nucleotides of 25S rRNA. The ability to produce fragment could not be separated from the ability of alpha sarcin to inhibit protein synthesis. Alpha sarcin also causes the specific cleavage of the 23S RNA of the E. coli subunit to produce a smaller fragment of RNA than that produced from eukaryote ribosomes.     

Two specific ribonucleoprotein fragments from rat liver 60S ribosomal subunits

K+-depleted 60S ribosomal subunits from rat liver were submitted to a mild treatment with ribonuclease T1. Ribonucleoprotein fragments could be separated on sucrose gradients only when the digested subunits were partially deproteinized with a high KCl concentration (0.6 M) which removed seven proteins more or less completely and 5S RNA. The RNA and protein content of each fragment has been characterized. The largest ribonucleoprotein enclosed two RNA fragments of about 950,000 and 750,000 daltons and all the salt-resistant proteins except L5. The smallest one enclosed protein L5 (with L11, L17 and L26 in small amounts) and a 67,000 RNA piece. The subsequent hydrolysis of the large ribonucleoprotein produced several other ribonucleoproteins. One of them has been fully characterized: it enclosed a 250,000 RNA fragment and protein L12 (with L11, L25 and L30 in smaller amounts).     

Cell-Free translation of Balbiani ring RNA (75S) ofChironomus tentans salivary glands into high molecular weight products

Summary Cell-free translation of salivary gland RNA or of purified Balbiani ring RNA (75S) in a reticulocyte lysate system gives rise to high molecular weight translational products (HMTP). In addition to a common size (approx. 1×10⁶ daltons) HMTP share imunogenic determinants with the giant secretory proteins of salivary glands. This suggests that HMTP correspond to in vivo secreted proteins and thus, corroborates the notion that 75S-RNA is the messenger for these proteins. The time course of HMTP synthesis and the lack of appearance of lower molecular weight components as translational products of 75S-RNA indicate that the synthesis of HMTP (and of secretory proteins) occurs in one piece by an uninterrupted process. HMTP are regarded the largest polypeptides so far synthesized in a cell-free system.     

Selektive Hemmung der Synthese der AMP-reichen RNS durch α-Amanitin in Zellen höherer Pflanzen

Summary The effect of -amanitin on the synthesis of AMP-rich RNA has been investigated. After incubation of freely suspended callus cells of parsley with the toxin and pulse labelling (30 min) with ³²P-orthophosphate, the high AMP content of the RNA component eluted from MAK columns behind the 25 S-RNA disappears. The base ratio of this RNA becomes ribosomal (CMP 20.1, AMP 26.5, GMP 28.4, UMP 25.0). Polyacrylamide gel electrophoresis of the high molecular RNA shows that radioactivity is incorporated only into the 32 S-RNA. At higher -amanitin concentrations the total nucleic acid synthesis is reduced. In this case only the high molecular RNA (32 S-RNA) is produced.     

Specificity of the amino acid transfer reaction inE. coli strains carrying different alleles of the RNA control (RC) gene

Summary The idea has been tested here that the aberration in amino acid controlled regulation of RNA synthesis in a mutant strain ofE. coli might reflect a major breakdown in the specificity of transfer of amino acids to S-RNA. For this purpose, S-RNA and amino acid activating enzymes were extracted from bacteria carrying either the normalRC ^st or the aberrantRC ^rel allele of the RNA control gene. The purified S-RNA preparations were first charged enzymatically with one or more of the 20 standard amino acids, then oxidized with periodate, and finally reisolated and retested for their residual capacity to accept an amino acid that was absent from the preliminary charging mixture. If preliminary charging transferred an amino acid to a non-cognate S-RNA species belonging to an absent amino acid, then the acceptor capacity for the missing amino acid would survive periodate oxidation and reveal its presence on recharging with that amino acid after post-periodate reisolation of the S-RNA. The results presented here show that there does not appear to exist any such major breakdown of transfer specificity in eitherRC ^st orRC ^rel bacteria: preliminary charging of the S-RNA fromRC ^rel bacteria with 19 of the 20 standard amino acids by use of the homologous amino acid activating enzymes does not afford protection against periodate oxidation for any appreciable fraction of the acceptor capacity for the absent 20th amino acid (when that amino acid is either methionine or arginine). It is unlikely, therefore, that thecatholic inducer, postulated to explain the continued RNA synthesis ofRC ^rel amino acid auxotrophs in the absence of their growth requirement, is one of the 20 standard amino acids.This investigation was supported by Public Health Service Research Grant CA 02129, from the National Cancer Institute.     

The primary and secondary structure of the 5'-end region of encephalomyocarditis virus RNA. A novel approach to sequencing long RNA molecules

The primary structure of a 149-nucleotide fragment of encephalomyocarditis (EMC) virus RNA from the 5'-terminus of the genome up to the poly(C) tract (S fragment) has been determined. For isolation of the S fragment, site-directed fragmentation of the viral RNA with RNase H and poly(dG) was employed. For sequencing the S fragment, a novel approach has been developed, which can be used for primary structure determination of long RNA molecules. A model of the secondary structure of the S fragment is proposed, according to which this region of RNA is highly structured. The role of complementary oligonucleotide stretches near both termini of the RNA molecule is discussed.     

Similarity of Vibrio alginolyticus, V. cholerae and other Vibrio species with respect to the structure of their flagellar apparatus and ribosomal 5S-RNA

Electron microscopic analysis of basal bodies of the flagella Vibrio alginolyticus revealed a structure composed of four discs. The diameters of two discs localized in the cytoplasmic membrane appeared to be twice as little as those of the other two discs. In this respect the basal body of V. alginolyticus resembles that of V. cholerae. The 5S sequence of ribosomal RNA from V. alginolyticus appeared to be similar to those of V. cholerae, V. harveyi and some other vibrios. Comparison of 5S-RNA sequence culminated in a dendrogram of evolutionary relationships of various bacterial species, suggesting that V. alginolyticus is a typical representative of the Vibrionacea family. The data obtained are discussed in terms of the role of Na+ energy metabolism in living cells.